A semi-automated LC–MS/MS method for the determination of LCI699, a steroid 11β-hydroxylase inhibitor, in human plasma

A novel liquid chromatographic method with tandem mass spectrometric detection (LC–MS/MS) for the determination of LCI699 was developed and validated with dynamic ranges of 0.0500–50.0 ng/mL and 1.00–1000 ng/mL using 0.0500 mL and 0.100 mL, respectively, of human plasma. LCI699 and the internal standard, [M + 6]LCI699, were extracted from fortified human plasma via protein precipitation. After transfer or dilution of the supernatant followed by solvent evaporation and/or reconstitution, the extract was injected onto the LC–MS/MS system. Optimal chromatographic separation was achieved on an ACE C18 (50 mm × 4.6 mm, 3 μm) column with 30% aqueous methanol (containing 0.5% acetic acid and 0.05% TFA) as the mobile phase run in isocratic at a flow rate of 1.0 mL/min. The total analysis cycle time is approximately 3.5 min per injection. The addition of an ion-pair reagent, TFA (0.05%, v/v), to the mobile phases significantly improved the chromatographic retention and resolution of the analyte on silica based reversed-phase column. Although addition of TFA to the mobile phase suppresses the ESI signals of the analyte due to its ion-pairing characteristics in the gas phase of MS source, this negative impact was effectively alleviated by adding 0.5% acetic acid to the mobile phase. The current method was validated for sensitivity, selectivity, linearity, reproducibility, stability and recovery. For the low curve range (0.0500–50.0 ng/mL), the accuracy and precision for the LLOQs (0.0500 ng/mL) were −13.0 to 2.0% bias and 3.4–19.2% CV, respectively. For other QC samples (0.100, 6.00, 20.0 and 40.0 ng/mL), the precision ranged from 1.2 to 9.0% and from 3.8 to 8.8% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from −11.3 to 8.0% and −7.2 to 1.6% bias, respectively, in the intra-day and inter-day batches. For the high curve range (1.00–1000 ng/mL), the accuracy and precision for the LLOQs (1.00 ng/mL) were 1.0–15.0% bias and 7.4–9.2% CV, respectively. For the other QC samples (3.00, 20.0, 200 and 750 ng/mL), the precision ranged from 0.8 to 7.0% and from 1.9 to 5.2% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from −2.5 to 4.0% and 0.7–1.0% bias, respectively, in the intra-day and inter-day batches. Additional assessments of incurred sample stability (ISS) and incurred sample reanalysis (ISR) were conducted to demonstrate the ruggedness and robustness of the assay method. The absence of adverse matrix effect and carryover was also demonstrated. The validated method was successfully used to support rapid turnaround human pharmacokinetic studies.